技术资料

It has been reported that human embryonic stem cells (hESCs) treated with BMP4 and inhibitors of TGF? signaling (A83-01) and FGF signaling (PD173074),called BAP,can efficiently differentiate to extraembryonic (ExE) cells in vitro. Due to restricted access to human embryos,it is ethically impossible to test the developmental potential of ExE cells in vivo. Here,we demonstrate that most ExE cells expressed molecular markers for both trophoblasts (TBs) and amniotic cells (ACs). Following intra-uterine transplantation,ExE cells contributed to the mouse placenta. More interestingly,ExE cells could chimerize with the mouse blastocyst as,after injection into the blastocyst,they penetrated its trophectoderm. After implantation of the injected blastocysts into surrogate mice,human cells were found at E14 in placental labyrinth,junction zones,and even near the uterine decidua,expressed placental markers,and secreted human chorionic gonadotropin. Surprisingly,ExE cells also contributed to cartilages of the chimeric embryo with some expressing the chondrogenic marker SOX9,consistent with the mesodermal potential of TBs and ACs in the placenta. Deleting MSX2,a mesodermal determinant,restricted the contribution of ExE cells to the placenta. Thus,we conclude that hESC-derived ExE cells can chimerize with the mouse blastocyst and contribute to both the placenta and cartilages of the chimera consistent with their heteogenious nature. Intra-uterus and intra-blastocyst injections are novel and sensitive methods to study the developmental potential of ExE cells. View Publication

Human GWAS have shown that obesogenic FTO polymorphisms correlate with lean mass,but the mechanisms have remained unclear. It is counterintuitive because lean mass is inversely correlated with obesity and metabolic diseases. Here,we use CRISPR to knock-in FTOrs9939609-A into hESC-derived tissue models,to elucidate potentially hidden roles of FTO during development. We find that among human tissues,FTOrs9939609-A most robustly affect human muscle progenitors’ proliferation,differentiation,senescence,thereby accelerating muscle developmental and metabolic aging. An edited FTOrs9939609-A allele over-stimulates insulin/IGF signaling via increased muscle-specific enhancer H3K27ac,FTO expression and m6A demethylation of H19 lncRNA and IGF2 mRNA,with excessive insulin/IGF signaling leading to insulin resistance upon replicative aging or exposure to high fat diet. This FTO-m6A-H19/IGF2 circuit may explain paradoxical GWAS findings linking FTOrs9939609-A to both leanness and obesity. Our results provide a proof-of-principle that CRISPR-hESC-tissue platforms can be harnessed to resolve puzzles in human metabolism. Human GWAS paradoxically linked FTO SNPs to both lean mass and sarcopenia/obesity. Here,Guang et al used CRISPR-edited stem cells to reveal that an obesogenic FTO SNP accelerates both muscle development and aging,by increasing RNA m6A demethylation. View Publication

Mutation in CUL4B gene is one of the most common causes for X-linked intellectual disability (XLID). CUL4B is the scaffold protein in CUL4B-RING ubiquitin ligase (CRL4B) complex. While the roles of CUL4B in cancer progression and some developmental processes like adipogenesis,osteogenesis,and spermatogenesis have been studied,the mechanisms underlying the neurological disorders in patients with CUL4B mutations are poorly understood. Here,using 2D neuronal culture and cerebral organoids generated from the patient-derived induced pluripotent stem cells and their isogenic controls,we demonstrate that CUL4B is required to prevent premature cell cycle exit and precocious neuronal differentiation of neural progenitor cells. Moreover,loss-of-function mutations of CUL4B lead to increased synapse formation and enhanced neuronal excitability. Mechanistically,CRL4B complex represses transcription of PPP2R2B and PPP2R2C genes,which encode two isoforms of the regulatory subunit of protein phosphatase 2 A (PP2A) complex,through catalyzing monoubiquitination of H2AK119 in their promoter regions. CUL4B mutations result in upregulated PP2A activity,which causes inhibition of AKT and ERK,leading to premature cell cycle exit. Activation of AKT and ERK or inhibition of PP2A activity in CUL4B mutant organoids rescues the neurogenesis defect. Our work unveils an essential role of CUL4B in human cortical development. View Publication

BackgroundAmyloid-beta (A?) plaques and their associated glial responses are hallmark features of Alzheimer’s disease (AD),yet their interactions within the human brain remain poorly defined.MethodsWe applied spatial transcriptomics (ST) and immunohistochemistry (IHC) to 78 postmortem brain sections from 21 individuals in the Religious Orders Study and Memory and Aging Project (ROSMAP). We paired ST with histological data and stratified spots into major categories of plaque-glia niches based on A?,GFAP,and IBA1 intensity. Leveraging published ROSMAP single-nucleus RNA-seq data,we examined differences in gene expression,cellular composition,and intercellular communication across these niches. Neuronal and glial changes were validated by IHC and quantitative analyses. We further characterized glial responses using gene set enrichment analysis (GSEA) with known mouse glial signatures and human AD-associated microglial states. Finally,we used iPSC-derived multicellular cultures and single-cell RNA sequencing (scRNA-seq) to identify cell types that,upon short-term A? exposure,recapitulate the glial responses observed in the human spatial data.ResultsLow-A? regions,enriched for diffuse plaques,exhibited transcriptomic profiles consistent with greater neuronal loss than high-A? regions. High-glia regions showed increased expression of inflammatory and neurodegenerative pathways. Spatial glial responses aligned with established gene modules,including plaque-induced genes (PIGs),oligodendrocyte (OLIG) responses,disease-associated microglia (DAM),disease-associated astrocytes (DAA),and human AD-associated microglial states,indicating that diverse glial phenotypes emerge around plaques and shape the local immune environment. IHC confirmed elevated neuronal apoptosis near low-A? plaques and greater CD68 abundance and synaptic loss near glia-high plaques. In vitro,iPSC-derived microglia—but not astrocytes—exposed to A? displayed transcriptomic changes that closely mirrored the glial states identified in our ST dataset.ConclusionsOur study provides a comprehensive spatial transcriptomic dataset from human AD brain tissue and bridges spatial gene expression with traditional neuropathology. By integrating ST,snRNA-seq,and human multicellular models,we map cellular states and molecular events within plaque-glia niches. This work offers a spatially resolved framework for dissecting plaque-glia interactions and reveals new insights into the cellular and molecular heterogeneity underlying neurodegenerative pathology.Supplementary InformationThe online version contains supplementary material available at 10.1186/s44477-025-00002-z. View Publication

AbstractA GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD),while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF,LTN1 and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter assays and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence,rather than amino acid content,is central to the impact of RQC factor depletion on RAN translation—suggesting a role for RNA secondary structure in these processes. Together,these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders. Graphical Abstract Graphical Abstract View Publication

The significant advances in the differentiation of human pluripotent stem (hPS) cells into pancreatic endocrine cells,including functional ?-cells,have been based on a detailed understanding of the underlying developmental mechanisms. However,the final differentiation steps,leading from endocrine progenitors to mono-hormonal and mature pancreatic endocrine cells,remain to be fully understood and this is reflected in the remaining shortcomings of the hPS cell-derived islet cells (SC-islet cells),which include a lack of ?-cell maturation and variability among different cell lines. Additional signals and modifications of the final differentiation steps will have to be assessed in a combinatorial manner to address the remaining issues and appropriate reporter lines would be useful in this undertaking. Here we report the generation and functional validation of hPS cell reporter lines that can monitor the generation of INS+ and GCG+ cells and their resolution into mono-hormonal cells (INSeGFP,INSeGFP/GCGmCHERRY) as well as ?-cell maturation (INSeGFP/MAFAmCHERRY) and function (INSGCaMP6). The reporter hPS cell lines maintained strong and widespread expression of pluripotency markers and differentiated efficiently into definitive endoderm and pancreatic progenitor (PP) cells. PP cells from all lines differentiated efficiently into islet cell clusters that robustly expressed the corresponding reporters and contained glucose-responsive,insulin-producing cells. To demonstrate the applicability of these hPS cell reporter lines in a high-content live imaging approach for the identification of optimal differentiation conditions,we adapted our differentiation procedure to generate SC-islet clusters in microwells. This allowed the live confocal imaging of multiple SC-islets for a single condition and,using this approach,we found that the use of the N21 supplement in the last stage of the differentiation increased the number of monohormonal ?-cells without affecting the number of ?-cells in the SC-islets. The hPS cell reporter lines and the high-content live imaging approach described here will enable the efficient assessment of multiple conditions for the optimal differentiation and maturation of SC-islets. View Publication

SummaryGene-switch techniques hold promising applications in contemporary genetics research,particularly in disease treatment and genetic engineering. Here,we developed a compact drug-induced splicing system that maintains low background using a human ubiquitin C (hUBC) promoter and optimized drug (LMI070) binding sequences based on the Xon switch system. To ensure precise subcellular localization of the protein of interest (POI),we inserted a 2A self-cleaving peptide between the extra N-terminal peptide and POI. This streamlined and optimized switch system,named miniXon2G,effectively regulated POIs in different subcellular localizations both in vitro and in vivo. Furthermore,miniXon2G could be integrated into endogenous gene loci,resulting in precise,reversible regulation of target genes by both endogenous regulators and drugs. Overall,these findings highlight the performance of miniXon2G in controlling protein expression with great potential for general applicability to diverse biological scenarios requiring precise and delicate regulation. Graphical abstract Highlights•miniXon2G is a compact and versatile version of the Xon gene-switch system•A P2A peptide eliminates residual peptides from functional proteins•We demonstrate applications on multiple proteins of interest•miniXon2G is a precise and reversible switch system with minimal background expression MotivationThe Xon drug-inducible splice-switch system is a simple and highly adaptable tool for regulated protein expression. We sought to further engineer this system to expand its applications in contemporary genetics research. In particular,we focused on reducing the size of the switch elements,maintaining minimal background expression,introducing a feature to remove extraneous peptide fragments,and demonstrating genomic integration and validation on a range of targets. Chi et al. develop a compact and versatile miniXon2G drug-inducible splice-switch system based on the Xon system. It features a reduced size,minimal background,and the removal of extraneous peptide fragments,enabling application to various biological scenarios that require precise expression control. View Publication

Cardiac differentiation of human induced pluripotent stem cells is readily achievable,yet derivation of mature cardiomyocytes has been a recognized limitation. Here,a mesoderm priming approach was engineered to boost the maturation of cardiomyocyte progeny derived from pluripotent stem cells under standard cardiac differentiation conditions. Functional and structural hallmarks of maturity were assessed through multiparametric evaluation of cardiomyocytes derived from induced pluripotent stem cells following transfection of the mesoderm transcription factor Brachyury prior to initiation of lineage differentiation. Transfection with Brachyury resulted in earlier induction of a cardiopoietic state as hallmarked by early upregulation of the cardiac-specific transcription factors NKX2.5,GATA4,TBX20. Enhanced sarcomere maturity following Brachyury conditioning was documented by an increase in the proportion of cells expressing the ventricular isoform of myosin light chain and an increase in sarcomere length. Mesoderm primed cells displayed increased reliance on mitochondrial respiration as determined by increased mitochondrial size and a greater basal oxygen consumption rate. Further,Brachyury priming drove maturation of calcium handling enabling transfected cells to maintain calcium transient morphology at higher external field stimulation rates and augmented both calcium release and sequestration kinetics. In addition,transfected cells displayed a more mature action potential morphology with increased depolarization and repolarization kinetics. Derived cells transfected with Brachyury demonstrated increased toxicity response to doxorubicin as determined by a compromise in calcium transient morphology. Thus,Brachyury pre-treatment here achieved a streamlined strategy to promote maturity of human pluripotent stem cell-derived cardiomyocytes establishing a generalizable platform ready for deployment. View Publication

ABSTRACTAutism spectrum disorder (ASD) and congenital heart disease (CHD) frequently co-occur,yet the underlying molecular mechanisms of this comorbidity remain unknown. Given that children with CHD are identified as newborns,understanding which CHD variants are associated with autism could help select individuals for early intervention. Autism gene perturbations commonly dysregulate neural progenitor cell (NPC) biology,so we hypothesized that CHD genes disrupting neurogenesis are more likely to increase ASD risk. Therefore,we performed an in vitro pooled CRISPR interference screen to identify CHD genes disrupting NPC biology and identified 45 CHD genes. A cluster of ASD and CHD genes are enriched for ciliary biology,and perturbing any one of seven such genes (CEP290,CHD4,KMT2E,NSD1,OFD1,RFX3 and TAOK1) impairs primary cilia formation in vitro. In vivo investigation of TAOK1 in Xenopus tropicalis reveals a role in motile cilia formation and heart development,supporting its prediction as a CHD gene. Together,our findings highlight a set of CHD genes that may carry risk for ASD and underscore the role of cilia in shared ASD and CHD biology. Highlighted Article: The increased likelihood of autism in individuals with congenital heart disease may stem from shared genetic mechanisms centered on cilia biology. View Publication

Rett syndrome (RTT) is a neurodevelopmental disorder that is caused by mutations in melty-CpG binding protein 2 (MeCP2). MeCP2 is a non-cell type-specific DNA binding protein,and its mutation influences not only neural cells but also non-neural cells in the brain,including vasculature associated with endothelial cells. Vascular integrity is crucial for maintaining brain homeostasis,and its alteration may be linked to the pathology of neurodegenerative disease,but a non-neurogenic effect,especially the relationship between vascular alternation and Rett syndrome pathogenesis,has not been shown. Here,we recapitulate a microvascular network using Rett syndrome patient-derived induced pluripotent stem (iPS) cells that carry MeCP2[R306C] mutation to investigate early developmental vascular impact. To expedite endothelial cell differentiation,doxycycline (DOX)-inducible ETV2 expression vectors were inserted into the AAVS1 locus of Rett syndrome patient-derived iPS cells and its isogenic control by CRISPR/Cas9. With these endothelial cells,we established a disease microvascular network (Rett-dMVNs) and observed higher permeability in the Rett-dMVNs compared to isogenic controls,indicating altered barrier function by MeCP2 mutation. Furthermore,we unveiled that hyperpermeability is involved in the upregulation of miR126–3p in Rett syndrome patient-derived endothelial cells by microRNA profiling and RNAseq,and rescue of miR126–3p level can recover their phenotype. We discover miR126–3p-mediated vascular impairment in Rett syndrome patients and suggest the potential application of these findings for translational medicine. View Publication

AbstractAimsDoxorubicin (DOX) is a widely used anthracycline anticancer agent; however,its irreversible effects on the heart can result in DOX-induced cardiotoxicity (DICT) after cancer treatment. Unfortunately,the pathophysiology of DICT has not yet been fully elucidated,and there are no effective strategies for its prevention or treatment. In this investigation,the novel role of transducin beta-like protein 1 (TBL1) in developing and regulating DICT was explored.Methods and resultsWe observed a reduction in TBL1 protein expression levels as well as cleavage events in the transplanted cardiac tissues of patients diagnosed with Dilated Cardiomyopathy and DICT. It was revealed that DOX selectively induces TBL1 cleavage at caspase-3 preferred sites—D125,D136,and D215. Interestingly,overexpression of the uncleaved TBL1 mutant (TBL1uclv) variant reduced apoptosis,effectively preventing DOX-induced cell death. We confirmed that cleaved TBL1 cannot form a complex with ?-catenin. As a result,Wnt reporter activity and Wnt target gene expression collectively indicate a decrease in Wnt/?-catenin signalling,leading to DICT progression. Furthermore,the cleaved TBL1 triggered DOX-induced abnormal electrophysiological features and disrupted calcium homeostasis. However,these effects were improved in TBL1uclv-overexpressing human-induced pluripotent stem cell-derived cardiomyocytes. Finally,in a DICT mouse model,TBL1uclv overexpression inhibited the DICT-induced reduction of cardiac contractility and collagen accumulation,ultimately protecting cardiomyocytes from cell death.ConclusionOur findings reveal that the inhibition of TBL1 cleavage not only mitigates apoptosis but also enhances cardiomyocyte function,even in the context of DOX administration. Consequently,this study's results suggest that inhibiting TBL1 cleavage may be a novel strategy to ameliorate DICT. Graphical Abstract Graphical Abstract View Publication

AbstractDespite the success of combination antiretroviral therapy (ART) for individuals living with HIV,mild forms of HIV-associated neurocognitive disorder (HAND) continue to occur. Brain microglia form the principal target for HIV infection in the brain. It remains unknown how infection of these cells leads to neuroinflammation,neuronal dysfunction,and/or death observed in HAND. Utilizing two different inducible pluripotent stem cell-derived brain organoid models (cerebral and choroid plexus [ChP] organoids) containing microglia,we investigated the pathogenic changes associated with HIV infection. Infection of microglia was associated with a sharp increase in CCL2 and CXCL10 chemokine gene expression and the activation of many type I interferon stimulated genes (MX1,ISG15,ISG20,IFI27,IFITM3 and others). Production of the proinflammatory chemokines persisted at low levels after treatment of the cell cultures with ART,consistent with the persistence of mild HAND following clinical introduction of ART. Expression of multiple members of the S100 family of inflammatory genes sharply increased following HIV infection of microglia measured by single-cell RNA-seq. However,S100 gene expression was not limited to microglia but was also detected more broadly in uninfected stromal cells,mature and immature ChP cells,neural progenitor cells and importantly in bystander neurons suggesting propagation of the inflammatory response to bystander cells. Neurotransmitter transporter expression declined in uninfected neurons,accompanied by increased expression of genes promoting cellular senescence and cell death. Together,these studies underscore how an inflammatory response generated in HIV-infected microglia is propagated to multiple uninfected bystander cells ultimately resulting in the dysfunction and death of bystander neurons. View Publication